The level of glycation of certain circulating proteins, e.g. hemoglobin, can be used to monitor the average blood glucose because glycation is a non-enzymatic, slow and continuous reaction that is primarily dependent on the ambient glucose concentration to which the hemoglobin is exposed during its residence time in the circulatory system. These two factors, glucose concentration and residence time, translate in vivo to the degree and duration of increased blood glucose concentration (hyperglycemia). Thus, when the blood glucose level is elevated, as it is in diabetic people whose diabetes is not well controlled, increased amounts of glycated hemoglobin are formed. The amount of glycated hemoglobin in an individual's blood reflects the average blood glucose concentration to which hemoglobin has been exposed during its life in the circulatory system. This period is about 100 days, so the determination of the concentration of glycated hemoglobin can provide an historical picture of the individual's blood glucose profile.
Methods described for the measurement of glycated hemoglobin include chromatography on an ion exchange column or boronate affinity columns, HPLC and agarose gel electrophoresis. Each of these techniques has drawbacks with respect to complexity, costly instrumentation, accuracy or variability.
Immuno techniques are available whereby monoclonal antibodies have been developed to react with epitopes of the glycated hemoglobin, i.e. that derivative of hemoglobin formed by the nonenzymatic reaction of glucose with reactive amine groups on the hemoglobin protein, and facilitate the determination of glycated hemoglobin concentration by standard immunochemistry techniques such as ELISA or a latex agglutination assay. Such an agglutination assay is disclosed in U.S. Pat. No. 4,970,171 wherein there is described an immunoassay for glycated hemoglobin, e.g. HbA1c, a variety of hemoglobin with glycation at the .beta. subunit in a blood sample, which involves the steps of:
a) treating the blood sample with a thiocyanate salt capable of denaturing the hemoglobin contained therein and an oxidant capable of converting the hemoglobin in the treated blood sample to the met-hemoglobin form; PA1 b) assaying the treated sample for met-hemoglobin as representing the amount of total hemoglobin in the sample; PA1 c) assaying the denatured, oxidized blood sample by immunoassay for the amount of denatured form of the particular hemoglobin derivative being sought; and PA1 d) calculating the relative amount of hemoglobin that is in the form of the hemoglobin derivative being sought compared to the total amount of hemoglobin in the test sample. PA1 a) assaying the blood sample for the total amount of hemoglobin present therein; PA1 b) assaying the blood sample for the amount of hemoglobin adduct present therein; PA1 c) normalizing the measurement from the hemoglobin adduct assay to the amount of total hemoglobin in the sample; and PA1 d) dividing the normalized hemoglobin adduct concentration by the total hemoglobin concentration to obtain the corrected concentration of hemoglobin adduct.
This glycated protein immunoassay was found to have a latent defect which affected its accuracy, which defect is based on the discovery that the response of a given concentration of glycated hemoglobin is not only dependent on its concentration but is also dependent on the total hemoglobin concentration. It has been discovered that there is an inverse relationship between the apparent glycated hemoglobin concentration and the total hemoglobin concentration, i.e. when the total hemoglobin concentration is high, the apparent glycated hemoglobin concentration is less than expected and when total hemoglobin concentration is low the apparent glycated hemoglobin concentration is higher than expected. The same phenomena is observed in the course of determining the concentration of other hemoglobin adducts by equilibrium analytical methods in which the equilibrium of the dimeric form and tetrameric form of the hemoglobin molecule is not perturbed by the chemistry. Immuno, enzymatic and chemical analytical methods are regarded as equilibrium methods whereas chromatographic and pepsin digestion methods are not. The present invention is predicated on the discovery of the underlying reason for this effect and the development of a method to overcome it.